One of the more debilitating skeletal manifestations of neurofibromatosis type 1 (NF1) is a tibial fracture that does not heal, known as pseudarthrosis (PA). Our pre-clinical studies have identified a therapeutic target that could potentially prevent the deficits in bone quality that lead to tibia fracture, but at present, there is no diagnostic tool to identify early enough those children with NF1 who are at risk of PA and who could benefit from treatment. There is thus a need for clinical instruments that are sensitive to important matrix characteristics (altered in NF1 bone disease) of cortical bone. Working toward the development of such instruments, the project will assess the ability of proton nuclear magnetic resonance relaxometry (NMR, which underpins MRI), Raman spectroscopy (RS), and cyclic reference point indentation (cRPI) to identify changes in bone quality following the loss of Nf1 in osteoprogenitors and importantly to explain the reduced stiffness and strength of Nf1-deficient bones. Each of these technologies are sensitive to different bone matrix characteristics: i) NMR measures matrix-bound water volume per bone volume (or as a concentration), ii) RS measures the amount of mineral relative to matrix content, secondary structure of collagen I, and the molecular order of the mineral crystals, and iii) RPI measures the resilience of the tissue matrix to micro-indentation and tissue stiffness. The utility of these measurements in predicting NF1 bone disease will be assessed in two pre- clinical studies involving the timed deletion of Nf1 in Osx-positive osteoprogenitors of Nf1 heterozygous mice (Nf1+/- in all other cells), thus mimicking what likely occurs in human NF1 bone disease. For Aim 1, long bones will be harvested from age-matched controls (Wild-type and Nf1+/- mice) and from the aforementioned mutant mice at different time points following Nf1 deletion upon removal of doxycycline (4, 8, and 16 weeks later). For Aim 2, controls and NF1 mutant mice will be treated with vehicle or Asfotase ?, a bone-targeting enzyme that hydrolyses the excess pyrophosphate generated following loss of Nf1 and promotes mineralization. Other mice will be treated with another FDA-approved drug Trametinib because MEK inhibition may also correct the mineralization defect. For both Aims, the femur and tibia mid-shafts will be imaged by micro-computed tomography to quantify structural properties and volumetric mineral density. After assessing bone quality characteristics with the matrix-sensitive techniques (NMR, RS, and RPI), the bones will be tested in three-point bending to measure structural rigidity and strength as well as estimated material modulus and strength. Using correlation analysis and general linear models, we will determine how well the matrix properties from each technique (plus combinations) explain the NF1-related decrease in fracture resistance and the treatment- related prevention of this decrease. Moreover, we will determine which matrix properties of bone quality are the first to change following Nf1 deletion and which are sensitive to treatment effects, thereby justifying clinical translation of NMR, RS, and/or RPI in NF1 disease as well as other genetic diseases affecting bone quality.